P605: T-CELL PROLYMPHOCYTIC LEUKEMIA: MOLECULAR CHARACTERIZATION OF A COHORT OF 18 PATIENTS AND EVALUATION OF BORTEZOMIB AS A POSSIBLE THERAPEUTIC STRATEGY
Authors: V. R. Gasparini, A. Martines, G. Barilà, A. Teramo, G. Calabretto, C. Vicenzetto, S. Carraro, V. Trimarco, L. Trentin, M. Facco, G. Semenzato, L. Bonaldi, R. Zambello
Journal: HemaSphere (2022)

Background: T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive mature T-cell malignancy characterized by a progressive clinical course and resistance to chemotherapy, whose pathogenesis is still largely unknown. Recurrent somatic mutations were detected within the JAK/STAT axis (i.e., JAK3 and STAT5b genes). T-PLL patients present with progressive lymphocytosis, splenomegaly and lymphadenopathy. New therapeutic strategies are needed to overcome the overall survival of two years observed in T-PLL patients even after treatment with alemtuzumab and consolidation with allogeneic stem cell transplantation. Ex-vivo drug sensitivity and resistance tests suggested proteasome inhibitors as novel candidates for the treatment of T-PLL. Aims: The investigation of the molecular features of T-PLL aimed at understanding whether JAK/STAT genetic lesions might have a role in the clinical management of patients. Ex-vivo treatment with Bortezomib (Bz), a proteasome inhibitor, was performed to test the affordability of this compound as a new option for T-PLL therapy. Methods: Sanger sequencing was performed on peripheral blood mononuclear cells (PBMC) of 18 T-PLL patients to detect the presence of somatic mutation in the hotspot regions of JAK3 and STAT5b genes. Patients were recruited at the Padova University Hospital. Immunophenotypic characterization of the leukemic clone was performed in all cases, while 11 patients underwent cytogenetic analysis upon mitogen stimulation. PBMC of 10 patients were cultured for 48h with different concentrations (1.3, 2.6, 5.2 and 7.8nM) of Bz according to literature. Annexin staining and western blot analyses were performed at both 24h and 48h to assess the efficacy of the treatment. Results: At least one mutation within the JAK/STAT pathway was detected in 39% (7/18) of patients. Consistent with the literature’s data, N642H variant was the most recurrent STAT5b mutation. Conversely, the most frequent variant in JAK3 gene was N564S, which has never been detected in T-PLL and not listed in the COSMIC database. Cytogenetic analysis revealed a complex karyotype in 9/11 (82%) patients with specific primary T-PLL aberrations: inv(14)(q11q32) or variant rearrangements in 78% (7/9) and t(X;14)(q28;q11) in 2 cases. Known secondary alterations such as those involving chromosome 8 and deletion of 11q22 were found in 89% (8/9) and 67% (6/9) of cases, respectively. Analysis of the clinical data did not reveal a correlation with the patient’s mutational status. Ex-vivo treatment with Bz induced apoptosis of the leukemic clone in a dose and time dependent manner. Compared to the untreated condition (16%), Bz concentration of 5.2nM increased the mortality of 1.7 (27%) and 4 (63.5%) times at 24h and 48h, respectively. The apoptosis was confirmed also at protein levels by a trend of caspase 9 and PARP cleavages upregulation at 48h. No difference in the sensitivity to treatment with Bz was observed between mutated and wild-type patients. Summary/Conclusion: No correlations between recurrent lesions within the JAK/STAT axis and clinical outcome were found. This points against a pathogenetic role of these mutations in T-PLL, conversely from other leukemias where STAT mutations are regarded as prognostic markers. This hypothesis is further sustained by the same response to the treatment with Bz. Since the treatment of T-PLL mostly remains an unmet clinical need, the finding of an increased apoptosis in Bz-treated cells paves the way for a deeper study on molecular mechanisms involved in the apoptosis of the leukemic clone.